The present invention relates to the detection of Herpes Simplex type 2 (HSV-2), more particularly, the invention relates to a transforming fragment of HSV-2 and to the detection thereof in clinical specimens.
Nearly one fifth of adults in the United States are infected with herpes simplex virus type 2 (HSV-2). Although HSV-2 is the most common cause of genital ulceration in developed countries, subclinical HSV-2 infections are suspected to affect a more important proportion of infected individuals. HSV-2 has also been proposed as a causative agent of genital cancer (Guibinga et al., 1995, Arch. STD/HIV Res. 9:163-179). However conflicting results from in vitro and in vivo studies have shed doubts on the role of this agent in cancer of the uterine cervix (Guibinga et al., 1995, Arch. STD/HIV Res. 9:163-179). A transforming region of the HSV-2 genomexe2x80x94the 7.6 kb BglII N (m.u. 0.58-0.63) segmentxe2x80x94has been identified, using transfection experiments. This was further supported by studies showing that BglII N sequences can also cooperate with oncogenic papillomas viruses to transform cells (DiPaolo et al., 1990, Virol. 777-779). Initially, the transforming ability of HSV-2 was thought to be located on the left-end (Xho-3 subfragment) of the BglII N segment (Galloway et al., 1983, Nature 392:21-24; Ibid., 1984, Proc. Natl. Acad. Sci. USA 81:4736-4740). However, neither the presence of a viral protein (Galloway et al., 1982, J. Virol. 42:530-537; Vonka et al., 1987, Adv. Cancer Res. 48:149-191) nor the persistence or integration (Galloway et al., 1983; Vonka et al., 1987) of specific HSV sequences, seemed to be required for the maintenance of the transformed phenotype (Pilon et al., 1989, Biochem. Biophys. Res. Comm. 159:1249-1261). The transforming ability of HSV-2 was left unexplained. Transfection of NIH 3T3 cells with the right-end (Kessous-Elbaz et al., 1989, J. Gen. Virol. 70:2171-2177; Pilon et al., 1989; Saavedra et al., 1985, EMBO J. 4:3419-3426) of the BglII N fragment (the Xho-1 and Xho-2 subfragments) showed an increase in the number of transformed foci, and HSV-2 sequences were retained more efficiently in transformed cells (Kessous-Elbaz et al., 1989; Pilon et al., 1989; Saavedra et al., 1985).
A number of clinical and epidemiologic studies have concluded that high risk papillomaviruses, such as HPV-16 and HPV-18 are necessary for the development of cervical cancer, but the long delay following infection indicates the importance of other factors (Kessler, 1986, In: Viral Etiology of Cervical Cancer, Peto et al., Eds. Cold Spring Harbor, N.Y., 55-64; and, zur Hauzen, 1989, Cancer Research 49:46774681), particularly other sexually transmitted infections (Kaufman et al., 1986, Clin. Obstet. Gynecol. 29:678-698; Macnab et al., 1989, Biomed. and Pharmacother. 43:167-172; zur Hausen, 1982, Lancet 2:1370-1372), for the development of malignancy. Although the etiologic link between herpes simplex virus-2 (HSV-2) and cervical cancer was proposed over two decades ago, the significance of the importance of HSV-2 to cervical cancer has been rather recent. The role for HSV-2 infection has been based primarily on sero-epidemiological data (Nahmias et al., 1970, Am. J. Epidemiol. 91:547-552; Rawls et al., 1968, Am. J. Epidemiol. 87:647-656) and on observation of viral antigens in exfoliated cells from patients with cervical dysplasia and cancer (Royston et al., 1970, Proc. Nat. Acad. Sci. 67:204-212). The difficulty in establishing a strong association was compounded by the lack of persistence of HSV sequences in the neoplastic cervical lesions (Macnab et al., 1989, Biomed. and Pharmacother. 43:167-172). In fact, in a prospective case-control study (Vonka, 1984, Int. J. Cancer 33:61-65) the investigators failed to observe such an association, which was later suggested may have resulted from overmatching of the cohort of women for sexual activity that minimized the risk factor (Reeves et al., 1989, New Engl. J. Med. 320:1437-1441). In other studies the lack of correlation of HSV-2 with cervical cancer was attributed to the use of immunoglobulin G instead of immunoglobulin A as a marker for the presence of HSV-2 (Corbino et al., 1989, Eur. J. Gynaecol. Oncol. 10:103-108).
A recent case-control study, using confirmed histological cases of cervical cancer from Latin America, found that the presence of HSV-2 antibodies correlated with a nine-fold excess risk of cervical cancer compared to women negative for HSV-2 or HPV-16/18 (Hildesheim et al., 1991, Int. J. Cancer 49:335-340). In well controlled studies of cervical cancer, others have reported a two-to-four fold excess risk in HSV-2 seropositive women (Slattery et al., 1989, Amer. J. Epidemiol. 130:248-258) and in women with both HPV and HSV-2 present in cervical tumor biopsies (Di Luca et al., 1989, Int. J. Cancer 43:570-577; Ibid., 1987, Int. J. Cancer 40:763-768.). Finally, in the last case control study (Daling et al., 1996, Cancer Epidemiology, Biomarkers and Prevention 5:541-548), involving women from western Washington state, the potential cofactors with HPVs in the development of cervical cancer were analysed. A significant increase in risk associated with HSV-2, as measured by antibodies, was found only in women whose tumor biopsies were negative for HPV. One major problem in establishing a definite link between HSV-2 and cervical cancer has been the difficulty to consistently detect HSV-2-specific DNA in cervical cancer biopsy samples despite the fact that several investigators have reported the presence of herpes virus specific sequences in some of the carcinomas tissues they have analysed (Frenkel et al., 1972, Proc. Nat. Acad. Sci. 69:3784-3789; Park et al., 1983, EMBO J. 2:1029-1034; Royston et al., 1970, Proc. Nat. Acad. Sci. 67204-212).
Experimental support for a role of HSV-2 in cervical cancer has come from in vitro studies that demonstrated its transforming potential using the inactivated virus (Duff et al., 1971, Nature 233:48-50; Macnab, 1974, J. Gen. Virol. 24:143-153) or its fragments, BglII N (mtr II) and BglII C (mtrIII) (Galloway et al., 1981, J. Virol. 38:749-760; Jariwalla et al., 1980, Proc. Natl. Acad. Sci. (USA) 77:2279-83; Reyes et al., 1979, Cold Spring Harbor Symp. Quant. Biol. 44:629-641). However the elucidation of the mechanism(s) leading to the transformed phenotype has been complicated by the loss of the viral sequences, which suggested the hypothesis of hit and run mechanism (Galloway et al., 1983, Nature 302:21-24) and review (Macnab et al., 1987, J. Gen. Virol. 68:2525-2550). Our studies on the transforming potential and the retention of BglII N and its Xhol restricted subfragments have suggested that BglII N, when present in its entirety, might have a toxic effect resulting from either a high copy number or from specific function(s) expressed by its coding sequences (Saavedra et al., 1985, EMBO J. 4:3419-3426). These studies also demonstrated that two BglII N subfragmentsxe2x80x94Xho-1+2 and Xho-2xe2x80x94induced the tumorigenic conversion of NIH3T3 cells and were stably retained in the transformed cell lines and their derived tumors (Saavedra et al., 1985, EMBO J. 4:3419-3426). The role of HSV-2 in cervical cancer has been further supported by in vitro studies showing its oncogenic cooperation with HPV 16/18. HPV-16 immortalized human foreskin keratinocytes transfected with a recombinant plasmid bearing the HSV-2 fragment BglII N yielded tumorigenic clones, whereas the parental HPV-immortalized cell lines were incapable of inducing tumors. Southern blot analysis of the viral sequences present in the transformed cell lines indicated that HPV-16 genomes were maintained unchanged in their integrated state, but HSV-2 sequences were not found in the tumor-derived cell lines (DiPaolo et al., 1990, Virology 177:777-779). Using a similar approach, Dhanwada et al. (Dhanwada et al., 1993, J. Gen. Virol. 74:955-963; Ibid., 1992, J. Gen. Virol. 73791-799) showed that HSV-2 mtrIII (BglII C) region induced rearrangements of HPV-18 DNA sequences in the immortalized human keratinocytes and chromosomal changes in HPV-16-immortalized human cell lines but did not produce the tumorigenic conversion observed with mtrII. Three of five HPV-18/HSV-2 cell lines retained the HSV-2 mtrIII DNA.
The sum of these recent clinical, epidemiological and experimental data is suggestive that HSV-2 might contribute to cervical cancer in women who are also infected with HPV-16/18. However, the exact role of herpes infection in the development of the cervical tumors still remains unclear.
There still remains a need to identify a molecular determinant of HSV-2 that cooperates with HPV to promote the development of cervical tumors. There also remains a need to identify the subfragment of HIV-2 BglII which could be responsible for the promotion of the tumorigenic conversion in human cervical cells. Finally, there remains a need to identify the role of HSV-2 BglII.
The diagnosis of HSV-2 infection is commonly performed using cell culture on appropriate clinical specimens (Gleaveset al., 1985, J. Clin. Microbiol. 21:29-32). However, the ability to isolate HSV-2 in cell culture is reduced in old lesions, in the presence of an host immune response and in episodes of reactivation (Lafferty et al., 1987, New Engl. J. Med. 316:1444-1449). In recent years, the use of the polymerase chain reaction (PCR) has allowed for the detection of HSV-2 DNA in diseases for which cell culture failed to establish a diagnosis, such as herpes simplex encephalitis (Kimura et al., 1991, J. Inf. Dis. 164:289-293; Lakeman et al., 1995, J. Inf. Dis. 171:857-863). Since it does not require the presence of infectious viral particles, PCR has also demonstrated the presence of viral DNA in culture-negative specimens (Cone et al., 1991, J. Inf. Dis. 164:757-760; Kimura et al., 1990, Med. Microbiol. Immunol. 179:77-84; Kriesel et al., 1994, J. Clin. Microbiol. 32:3088-3090; Nahass et al., 1992, JAMA 268:2541-2544; Rogers et al., 1992, Obstet. Gynecol. 79:464-469). PCR (or other amplification methods) could thus prove useful for the analysis of specimens sent to the laboratory from remote clinics. PCR (or other amplification methods) could also identify asymptomatic shedding of HSV-2 (Hardy et al., 1990, J. Inf. Dis. 162:1031-1035), a clinical situation where the titer of HSV is reduced (Corey et al., 1988, Microbiol. Infect. Dis. 4 (Suppl.): 111S-119S). PCR assays amplifying sequences from the left-end of BglII N have been described (Lulitanond et al., 1994, Mol. Cell Probes 8:441-447; Yamakawa et al., 1994, APMIS 102:401-406). These studies demonstrated that in opposite to normal tissue, precancerous of cancerous lesions of the uterine cervix contained specific HSV-2 DNA sequences. However, the use of the left-end of BglII N does not provide the sensitivity required for proper detection of HSV-2 infection in uterine cervix cancer. Indeed, the left-end of BglII N is often lost in these cancer cells, thereby leading to false negative results.
There thus remains a need for the development and optimization of an assay, for the specific detection in clinical specimens of HIV-2 specific sequences. There also remains a need for a tool for the specific detection of transforming sequences of HSV-2 from the right end of BglII N and more generally for the typing of the HSV infection in question.
The invention provides, in general, isolated nucleic acid molecules coding for Xho-2 or fragments thereof.
The invention further provides purified Xho-2 polypeptides or an epitope binding portion thereof.
The invention also provides nucleic acids for the specific detection of the presence of nucleic acids encoding Xho-2 proteins or polypeptides in a sample.
The invention further provides a method of detecting nucleic acid encoding Xho-2 in a sample.
The invention also provides a kit for detecting the presence of nucleic acid encoding Xho-2 in a sample.
The invention further provides a recombinant nucleic acid molecule comprising, 5xe2x80x2 to 3xe2x80x2, a promoter effective to initiate transcription in a host cell and the above-described isolated nucleic acid molecule.
The invention also provides a recombinant nucleic acid molecule comprising a vector and the above-described isolated nucleic acid molecule.
The invention further provides an antisense Xho-2 nucleic acid molecule.
The invention also provides a cell that contains the above-described recombinant nucleic acid molecule.
The invention further provides a non-human organism that contains the above-described recombinant nucleic acid molecule.
The invention also provides an antibody having binding affinity specifically to Xho-2 or an epitope-bearing portion thereof.
The invention further provides a method of detecting Xho-2 in a sample.
The invention also provides a method of measuring the amount of Xho-2 in a sample.
The invention further provides a method of detecting antibodies having binding affinity specifically to Xho-2.
The invention further provides a diagnostic kit comprising a first container means containing the above-described antibody, and a second container means containing a conjugate comprising a binding partner of the monoclonal antibody and a label.
The invention also provides a hybridoma which produces the above-described monoclonal antibody.
The invention further provides diagnostic methods for human disease, in particular, genital cancer. Preferably, a method of diagnosing the presence or predisposition to develop genital cancer in a patient is provided herein.
The invention also provides methods for therapeutic uses involving all or part of (1) a nucleic acid sequence encoding Xho-2, (2) antisense Xho-2 nucleic acid molecules, (3) Xho-2 protein, or (4) Xho-2 antibodies.
Further objects and advantages of the present invention will be clear from the description that follows.
More particularly, this invention concerns the cloning and nudeic acid sequencing of the Xho-2 subfragment of BglII N, located at the right end of BglII N of HSV-2 and to probes derived therefrom. From this sequence, primers that specifically detect sequences from this region were identified. These nucleic acid sequences can be used in diagnostic procedures, to identify the type of HSV present in a given biological sample. Further, these nucleic acid sequences provide the additional advantage of permitting the identification of the HSV-2 molecular determinant responsible for the transformation activity of HSV-2 in vivo, the right end of BglII N. The invention also concerns methods using these nucleic acid sequences as well as kits comprising same.
The invention also relates to an optimized and validated assay for the investigation of the role of HSV-2 as a cofactor in genital cancer, more specifically, cervical cancer. It also relates to the development and optimization of an amplification assay for the specific detection in clinical specimens of transforming sequences of HSV-2 from the right end of BglII N. In a preferred embodiment, it relates to a PCR assay therefor. Various clinical isolates of herpes viruses were evaluated in vitro in this PCR assay. The optimized PCR assay was then compared with cell culture for the detection of HSV-2 infection in specimens collected from various sites and submitted to the diagnostic laboratory for viral culture.
The invention also relates to the Xho-2 fragment having a sequence of 2480 bp and encoding an open reading frame (ORF) specifying a putative protein of 413aa. The invention thus also refers to the Xho-2 protein, antibodies thereto and to ligands which specifically bind thereto. In a related aspect, the invention also relates to the use of the Xho-2 nucleic acid or protein sequence or parts or derivatives thereof to transform an immortalized cell. Therefore, the present invention relates to a model for the analysis of the contribution of HSV-2 complete or potential sequences to cervical cancer and more particularly, to its complementation with HPV.
In addition, the present invention relates to the Xho-2 nucleic acid sequence, Xho-2 protein fragments or derivatives thereof as targets for a diagnosis of HSV-2 presence and potentially of prognosis of cervical cancer.
The applicant has determined the sequence of the Xho-2 fragment of BlgII N. Having identified regions of HSV-2 Xho-2 which are specific thereto, the applicant is the first to provide a means of obtaining diagnostic tools for the typing of HSV viruses. By demonstrating that Xho-2 has a transforming capacity or oncogenic function in HPV-immortalized cervical cells, the applicant is thus the first to have identified the HSV-2 molecular determinant responsible for the transformation activity of HSV-2 in genital cancer. In so doing, the applicant is the first to have derived a cellular model of cervical cancer. The applicant has also derived three probes from the Xho-2 nucleic acid sequence which enable a sensitive (down to 10 copies and even less in a preferred embodiment) and specific (without cross-reactivity with other HSV, human genomic sequence or HPV) assay for the detection and identification of HSV-2 sequences in a sample such as a biological sample.
It will be-clear to a person of ordinary skill, to which this application pertains, that in the context of a diagnostic test, the nucleic acid sequences of the present invention, although preferably used in the context of an amplification method which permits an increase in sensitivity of detection and identification, can also be used in hybridization experiments such as, for example, Southern blots or Northern blots.
It should be noted that because of the G-C content, the obtention of probes or primers, which display the sensitivity and specificity (HSV-2-specific) of those described herein, is complexified. However, by providing the nucleic acid sequence of Xho-2, the applicant enables the design of additional primer pairs or probes which could demonstrate the same level of sensitivity and specificity as probes disclosed hereinbelow. Additional probes or primers displaying adequate specificity and sensitivity can be obtained in accordance with the present invention or in accordance with other well known methods. These probes and primers could be tested in accordance to the present invention and according to known methods to verify their specificity and sensitivity.
It will also be understood that the use of PCR technology to amplify the targeted HSV-2 sequence is only a preferred method of amplification, as other methods are well known to a person of ordinary skill (see below). Accordingly, the present invention cannot be limited to a particular type of amplification method.
Nucleotide sequences are presented herein by single strand only, in the 5xe2x80x2 to 3xe2x80x2 direction, from left to right. One letter nucleotide symbols used herein have their standard meaning in the art in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission.
In the description that follows, a number of terms used in recombinant DNA (rDNA) technology are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
Isolated Nucleic Acid Molecule: An xe2x80x9cisolated nucleic acid moleculexe2x80x9d, as is generally understood and used herein, refers to a polymer of nucleotides, and includes but should not be limited to DNA and RNA. The xe2x80x9cisolatedxe2x80x9d nucleic acid molecule is purified from its natural in vivo state.
Recombinant DNA: Any DNA molecule formed by joining DNA segments from different sources and produced using recombinant DNA technology (a.k.a. molecular genetic engineering).
DNA Segment: A DNA segment, as is generally understood and used herein, refers to a molecule comprising a linear stretch of nucleotides wherein the nucleotides are present in a sequence that can encode, through the genetic code, a molecule comprising a linear sequence of amino acid residues that is referred to as a protein, a protein fragment or a polypeptide.
The term xe2x80x9camplification pairxe2x80x9d: As used herein, refers to a pair of oligonucleotides of the present invention selected to be suitable for use together in amplifying a selected HSV-2 nucleic acid sequence by an amplification process such as polymerase chain reaction, ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below.
Nucleic acid (i.e., DNA or RNA) samples for practicing the present invention may be obtained from any suitable source. Typically, the nucleic acid sample will be obtained in the form of a clinical sample of a biological fluid or biological tissue to be assessed as containing the HSV-2 sequences. It will be apparent that the present invention also permits the detection of HSV-2 nucleic acid sequences in non-clinical samples. Suitable clinical samples include, but are not limited to, genital and non-genital samples.
Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format employed. In general, the oligonucleotide probes or primers are at least 15 nucleotides in length. For example, oligonucleotide probes or primers used for detecting HSV-2 are preferably about 20 nucleotides in length. The oligonucleotide probes or primers may be adapted to be especially suited to a chosen nucleic acid amplification system.
Nucleic Acid Hybrdization: Nudeic add hybridization depends on the principle that two single-stranded nucleic acid molecules that have complementary base sequences will reform the thermodynamically favored double-stranded structure if they are mixed under the proper conditions. The double-stranded structure will be formed between two complementary single-stranded nucleic acids even if one is immobilized on a nitrocellulose filter. In the Southern hybridization procedure, the latter situation occurs. The DNA of the individual to be tested can be digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, converted to the single-stranded form, and transferred to nitrocellulose paper, making it available for reannealing to the hybridization probe. Examples of hybridization conditions can be found in Ausubel, F. M. et al., Current protocols in Molecular Biology, John Wily and Sons, Inc., New York, N.Y. (1989). A nitrocellulose filter is incubated overnight at 68xc2x0 C. with labeled probe in a solution containing 50% formamide, high salt (either 5xc3x97SSC[20xc3x97: 3M NaCl/0.3M trisodium citrate] or 5xc3x97SSPE [20xc3x97: 3.6M NaCl/0.2M NaH2PO4/0.02M EDTA, pH 7.7]), 5xc3x97Denhardt""s solution, 1% SDS, and 100 xcexcg/ml denatured salmon sperm DNA This is followed by several washes in 0.2xc3x97SSC/0.1% SDS at a temperature selected based on the desired stringency: room temperature (low stringency), 42xc2x0 C. (moderate stringency) or 68xc2x0 C. (high stringency). The temperature selected is determined based on the melting temperature (Tm) of the DNA hybrid.
Stringent conditions (e.g., conditions represented by a wash stringency of 0.5xc3x97SSC and 0.1% SDS at a temperature of 20 or 30 degrees below the melting temperature of the probe or even conditions represented by a wash stringency of 0.1xc3x97SSC and 0.1% SDS at a temperature of 10 degrees below the melting temperature of the duplex of probe and target nucleic acid in a standard hybridization assay) will be preferably used; see J. Sambrook et al., Molecular Cloning, A Laboratory manual, 2d Ed. 1989, Cold Spring Harbor Laboratory.
Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and xcex1-nucleotides and the like. Modified sugar-phosphate backbones are generally illustrated by Miller ,1988, Ann. Reports Med. Chem. 23:295 and Moran et al., 1987, Nucleic acid molecule. Acids Res., 14:5019. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), with DNA preferred.
Use of probes in detection methods include Northern blots (RNA detection), Southern blots (DNA detection), western blots (protein detection), and dot or slot blots (DNA, RNA or protein). Other detection methods include kits containing probes on a dipstick setup and the like.
Hybrid molecules formed from using the probes of the invention can be detected by using a detectable marker which is added to one of the probes. Probes can be labeled by several methods. Probes can be radiolabeled and detected by autoradiography. Such labels for autoradiography include 3H, 125I, 35S, 14C, and 32P. Typically, the choice of radioactive isotopes depends on research preferences involving ease of synthesis, stability, and half lives of the isotopes. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, electrochemical via sensors, time-resolved fluorescence, enzymes, and antibodies. For example, an antibody can be labeled with a ligand. Other detectable markers for use with probes of the invention include biotin, radionucdeotides, enzyme inhibitors, co-enzymes, luciferins, paramagnetic metals, spin labels, and monoclonal antibodies. The choice of label dictates the manner in which the label is bound to the probe.
Radioactive nucleotides can be incorporated into probes of the invention by several means. Such means include nick translation of double-stranded probes, copying single-stranded M13 plasmids having specific inserts with the Klenow fragment of DNA polymerase I of E. coli or other such DNA polymerase in the presence of radioactive dNTP, transcribing CDNA from RNA templates using reverse transcriptase in the presence of radioactive dNTP, transcribing RNA from vectors containing strong promoters such as SP6 promoters or T7 promoters using SP6 or T7 RNA polymerase in the presence of radioactive rNTP, tailing the 3xe2x80x2 ends of probes with radioactive nucleotides using terminal transferase, and by phosphorylation of the 5xe2x80x2 ends of probes using gamma 32P ATP and polynucleotide kinase.
Oligonucleotide or Oligomer: A molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. An oligonucleotide can be derived synthetically or by cloning.
Amplification Primer: An oligonucleotide which is capable of annealing adjacent to a target sequence and serving as an initiation point for DNA synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is initiated.
Amplification of a selected, or target, nucleic acid sequence may be carried out by any suitable means. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25. Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction, ligase chain reaction, strand displacement amplification, transcription-based amplification (see Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177), self-sustained sequence replication (see Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87, 1874-1878), the Qxcex2 replicase system (see Lizardi et al., 1988, BioTechnology 6:1197-1202) and NASBA (Malek et al., 1994, Methods Mol. Biol., 28:253-260). Preferably, amplification will be carried out using PCR.
Polymerase chain reaction (PCR) is carried out in accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188. In general, PCR involves, first, treating a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) with one oligonucleotide primer for each strand of the specific sequence to be detected under hybridizing conditions. An extension product of each primer which is synthesized is complementary to each nucleic acid strand, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith. The extension product which is synthesized from each primer, when separated from its complement, can then serve as a template for the synthesis of the extension product of the other primer. The sample is then treated under denaturing conditions to separate the primer extension products from their templates and the sample analyzed to assess whether the sequence or sequences to be detected are present. These steps are cyclically preferred until the desired degree of amplification is obtained. Detection of the amplified sequence may be carried out by adding to the reaction product an oligonucleotide probe capable of hybridizing to the reaction product (e.g. an oligonucleotide probe of the present invention), the probe carrying a detectable label, and then detecting the label in accordance with known techniques.
Ligase chain reaction (LCR) is carried out in accordance with known techniques (Weiss, 1991, Science 254:1292. Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill. Strand displacement amplification (SDA) is also carried out in accordance with known techniques or adaptations thereof to meet the particular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA 89:392-396; and Ibid., 1992, Nucleic Acids Res. 20:1691-1696.
Vector: A plasmid or phage DNA or other DNA sequence into which DNA of the present invention can be inserted to be cloned. The vector can replicate autonomously in a host cell, and can be further characterized by one or a small number of endonuclease recognition sites as well as preferably a marker suitable for use in the identification of cells transformed with the vector. The words xe2x80x9ccloning vehiclexe2x80x9d are sometimes used for xe2x80x9cvector.xe2x80x9d
Expression: Expression is the process by which a structural gene produces a polypeptide. It involves transcription of the gene into mRNA, and the translation of such mRNA into polypeptide(s).
Expression Vector: A vector or vehicle similar to a cloning vector but which is capable of expressing a gene which has been cloned into it, after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.
Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
Functional Derivative: A xe2x80x9cfunctional derivativexe2x80x9d of a sequence, either protein or nucleic acid, is a molecule that possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of the protein or nucleic acid sequence. A functional derivative of a protein can contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function. The term xe2x80x9cfunctional derivativexe2x80x9d is intended to include the xe2x80x9cfragments,xe2x80x9d xe2x80x9csegments,xe2x80x9d xe2x80x9cvariants,xe2x80x9d xe2x80x9canalogs,xe2x80x9d or xe2x80x9cchemical derivativesxe2x80x9d of a molecule.
As used herein, a molecule is said to be a xe2x80x9cchemical derivativexe2x80x9d of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule""s solubility, absorption, biological half life, and the like. The moieties can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like. Moieties capable of mediating such effects are disclosed in Remington""s Pharmaceutcal Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art.
Variant: A xe2x80x9cvariantxe2x80x9d of a protein or nucleic acid is meant to refer to a molecule substantially similar in structure and biological activity to either the protein or nucleic acid. Thus, provided that two molecules possess a common activity and can substitute for each other, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical.
Allele: An xe2x80x9callelexe2x80x9d is an alternative form of a gene occupying a given locus on the chromosome.
Mutation: A xe2x80x9cmutationxe2x80x9d is any detectable change in the genetic material which can be transmitted to daughter cells and possibly even to succeeding generations giving rise to mutant cells or mutant individuals. If the descendants of a mutant cell give rise only to somatic cells in multicellular organisms, a mutant spot or area of cells arises. Mutations in the germ line of sexually reproducing organisms can be transmitted by the gametes to the next generation resulting in an individual with the new mutant condition in both its somatic and germ cells. A mutation can be any (or a combination of) detectable, unnatural change affecting the chemical or physical constitution, mutability, replication, phenotypic function, or recombination of one or more deoxyribonucleotides; nucleotides can be added, deleted, substituted for, inverted, or transposed to new positions with and without inversion. Mutations can occur spontaneously and can be induced experimentally by application of mutagens. A mutant variation of a nucleic acid molecule results from a mutation. A mutant polypeptide can result from a mutant nucleic acid molecule.
Purified: A xe2x80x9cpurifiedxe2x80x9d protein or nucleic acid is a protein or nucleic acid that has been separated from a cellular component. xe2x80x9cPurifiedxe2x80x9d proteins or nucleic acids have been purified to a level of purity not found in nature.
Substantially Pure: A xe2x80x9csubstantially purexe2x80x9d protein or nucleic acid is a protein or nucleic acid preparation that is lacking in all other cellular components.
A kit for detecting HSV-2 nucleic acid in a clinical sample contains at least one nucleic acid sequence of the present invention, and hybridization solution for enabling hybridization between this sequence and the nucleic acid sample, with the nucleic acid sequence either suspended in the solution or provided separately in lyophilized form. One example of a suitable hybridization solution is a solution comprised of 6xc3x97SSC (0.9M sodium chloride, 0.09M sodium citrate, pH7.0), 0.1M EDTA pH 8.0, 5xc3x97Denhardt""s solution [0.1% (w/v) Ficoll(trademark) Type 400, 0.1% (w/v) polyvinylpyrrolidone, 0.1% (w/v) bovine serum albumin], and 100 xcexcg/ml sheared, denatured salmon sperm DNA, commercially available from Bethesda Research Laboratories, Gaithersburg, Md. 20877 USA under Catalog No. 5565UA. See also Sambrook et al., 1989, A Laboratory Manual, 2nd Edition, 387-388, Cold Spring Harbor Laboratory. For example, the components of the kit are packaged together in a common container (e.g., a container sealed with a frangible seal), the kit typically including an instruction sheet for carrying out a specific embodiment of the method of the present invention. Additional optional components of the kit, depending on the assay format to be employed, include a second nucleic acid sequence of the invention suitable for use with the first sequence for carrying out PCR as explained above (or, in the case of a kit for carrying out LCR, two pairs of probes or primers of the present invention), none, one or more detection probes, and means for carrying out a detecting step (e.g., a probe or primer of the invention labelled with a detectable marker and optionally an enzyme substrate when the detectable marker is an enzyme).
It should be understood that having now recognized that the Xho-2 nucleic acid segment is transforming in the contact of an HPV-immortalized cervical cell, and hence it can complement HPV and transform same, inhibitors of the Xho-2 transforming activity could be screened and identified using the genital cancer cell line of the invention.
By xe2x80x9cantisense moleculesxe2x80x9d is meant nudeic acid fragments which are complementary to their target and eventually lead an inhibition of the production of the protein encoded by this target. These antisense can be small segments of nucleic acids or long ones. They can also comprise modifications which enhance their stability. The design of appropriate antisense molecules and derivatives thereof is well known to an artisan of ordinary skill. These antisense against Xho-2 can be tested for their effects on the transforming activity of Xho-2 in accordance with the present invention or other suitable methods. In one embodiment, the full antisense Xho-2 nucleic acid sequence could be used. In a preferred embodiment, complementary regions to the coding sequence of Xho-2 could be used as antisense.